Effects of 17alpha-methyltestosterone exposure on steroidogenesis and cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua)

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.